The objective of this proposal is to understand the basis for the switch from fetal to adult hemoglobin. Because of the complexity of the eukaryotic genome, the mechanism of the switch has been difficult to study using whole cells, nuclei, or chromatin. For this reason, and because the study of regulation in bacterial cells has been greatly aided by the purification of specific genes, our approach is to purify small amounts of the globin genes by chemical procedures and then prepare large quantities of these genes by recombinant cloning techniques. This DNA can then be used to study the sequence organization of the globin genes and to investigate in vitro factors which affect their expression. As a globin switch similar to that found in humans appears in goats, they will be used as the experimental animals. Methods utilizing both R-Loop hybridization and selection of specific restriction fragments will be used in the purification. The first method involves hybridization of mercurated globin mRNA to high molecular weight whole cell DNA unde R-Loop conditions. The DNA containing the globin mRNA hybrid is then isolated from the bulk of the DNA by chromatography on sulfhydryl agarose. Separation of DNA restriction fragments containing globin genes will also be used to prepare DNA enriched in these genes. Once the DNA is highly purified it will be used to form recombinant molecules with the plasmid pMB9 and used to transfect Curtiss E. coli strain 1776. Hybridization probes for detecting the globin genes in cells cloned from transfected cells will be prepared from the various globin mRNAs (alpha, gamma, beta A, beta D (E)) by plasmid cloning of cDNA duplexes prepared from the mRNAs. When globin gene containing DNA sequences have been isolated from the plasmid cloned material restriction endonuclease mapping will be done to determine the sequence organization and structural similarities among the various globin genes. Ultimately, we plan to determine the nucleotide sequence of the DNA surrounding the globin genes, particularly at the 5' end with the hope of elucidatng possible regulatory or promotor sites and to investigate the factors which regulate the transcription of the globin genes in vitro.